This invention involves a new kind of triplex forming oligonucleotide (TFO), particularly a new triplex forming oligonucleotide and their derivatives that form triplex DNA with two similar homopoly purine and pyrimidine fragments. This invention also involves the application of TFO in inhibiting the hepatitis B virus.
In the 1980s, it was found that in vivo double stranded DNA (dsDNA) formed by homopolypurine/homopolypyrimidine sequences can fold over, and one of the strands can then form triplex DNA with the 5xe2x80x2-upstream homopolypurine/homopolypyrimidine sequence of dsDNA. The other strand then becomes single stranded DNA (ssDNA). Furthermore, the triplex DNA can become involved in the regulation of gene expression (Lyamichev V I, Mirkin S M, et al., J Biomol Struct Dyn, 1986, 3: 667-9; Larsen A, Weintraub H et al., Cell, 1982, 29: 609-22; Htun H, Dahlberg J E, Science, 1989, 243: 1571-76). Triplex DNA can be also be formed by the in vitro recognition of oligonucleotides with specific target DNA. A strand of homopolypurine/homopolypyrimidine oligonucleotides can specifically recognise a complementary sequence of homopolypurines/homopolypyrimidines in dsDNA by base pair complementation, and can form a stable triplex DNA structure by linking with purine chains in dsDNA via Hoogsteen bonds or anti-Hoogsteen bonds. The oligonucleotide fragment that forms triplex DNA with dsDNA is named Triplex Forming Oligonucleotide (TFO). The formation of triplex DNA can inhibit the binding of proteins to DNA. TFO carrying a terminal EDTA-Fe2+ to bind a specific target sequence can form triplex DNA, resulting in the targeted dsDNA being cleaved at that specific position in the triplex (Moser H. E., Dervan P. B., Science, 1987, 238: 645-50). Therefore, it is possible to inhibit gene expression at the DNA level by the formation of triplex DNA, which gives rise to the so-called Antigene Strategy.
This technology is superior to anti-sense and nuclease technologies. TFO uses a specific sequence of dsDNA as a binding site to form triplex DNA or to cleave targeted DNA at a specific location in the triplex DNA. This inhibits transcription or gene replication. Anti-sense nucleic acids and nuclease enzymes both target mRNA. They cause the inhibition of gene expression by binding with mRNA to stop translation, or by promoting the degradation of mRNA. In cells, a single copy of DNA can produce multiple copies of mRNA. Therefore, it may be more efficient at the DNA level to block gene replication or transcription. DNA replication or RNA transcription can be inhibited by two strategies based on the theory of triplex DNA formation and the rules of base pair complementarity. One strategy is to use a TFO fragment based on a promoter of a particular gene, so that it forms triplex DNA by binding to the complementary sequence of the targeted gene, thereby blocking the binding of protein to DNA. In the second strategy, a TFO fragment is designed based on a specific portion of a gene, with which it forms triplex DNA by binding to the complementary sequence of the targeted gene. This inhibits DNA replication or RNA transcription by blocking the movement of the replication-transcription complex. In 1988, Cooney and coworkers proved that the molecular-triplex DNA structure formed at the starting point of c-myc inhibits the transcription of c-myc (Cooney M., Science, 1988, 241:450-9). However, up until now, there have been very few practical applications of TFO in the inhibition of gene expression. In a review of international patents, only one such patent has been found, and this involved the application of TFO to inhibit the expression of androgen receptor gene (U.S Pat. No. 5,556,956, Sep. 17, 1996). The main reason for this lack of applications is that it is very rare to find in promoters or other particular regions of genes homopolypurine/homopolypyrimidine sequences which are long enough. The triplex DNA formed by relatively short TFOs and target dsDNA is not particularly stable or specific, therefore their ability to act as inhibitors is weak, which limits their practical application in the Antigene Strategy. There have only been a few theoretical studies on the range of target dsDNA that can form triplex DNA, but no practical applications have resulted. Horne and Dervan et al designed an alternating triplex DNA containing two fragments of homopolypurine sequences located on the two chains of dsDNA, whereby a part of the TFO sequence matches a purine chain in the double strand, and the other part of the TFO matches the other purine chain in the double strand (Home D A, Dervan P B, J. Am. Chem. Soc., 1990; 112: 2435-37).
The stability of triplex DNA has also been studied. It was found that triplex DNA could still be formed even though it contains a single mismatched base, but its stability is markedly decreased. Therefore, problems are still to be resolved in finding new TFO structures that can be used to inhibit the expression of harmful genes. There have also been studies on increasing the stability and inhibitory effect of TFO by chemical modification, including thiophosphate modification (Tu, et al., J. Biol. Chem., 1995, 270: 28402-7), a single amino acid linked to the 3xe2x80x2-end (Orsen F. M., et al. Nucleic Acids Res. 1991, 19: 3435-41; Postel E. H., et al. Proc. Natl. Acad. Sci. USA 1991, 88: 8827-31; McShan W. M., et al. J. Biol. Chem. 1992, 267: 5712-21), cholesterol linked to the 3xe2x80x2-end (Ing N. H. et al., Nucleic Acids Res., 1993, 21: 2789-96) and so on. Although the stability and inhibitory capability of TFO can be increased by chemical modification, the length of TFO is still the key factor in TFO stability.
HBV is a hepatic DNA virus that can cause acute and chronic hepatitis. Eighty percent of patients with Hepatocellular Carcinoma (HCC) have HBV infection. The relative risk of HCC in the population with chronic HBV infection increases at least 100 fold. China is a high epidemic region for hepatitis B. Eight to 10% (100 million) of the population are positive to Hepatitis B (virus) Surface Antigen (HBsAg). Hepatitis B caused by HBV and the associated HCC are one of the major health issues in the world. However, as of the present, there is still no effective therapeutic regimen in the clinic. Hence the development of TFO against HBV as a new therapeutic approach is of contemporary interest. However, as the HBV genes do not contain homopolypurine/homopolypyrimidine sequences which are long enough to be a practicable target for TFO, it is worthwhile to search for a new triplex DNA structure.
The aim of the invention is to provide a type of TFO which is able to form a triplex DNA structure with two fragments of homologous homopolypurine/homopolypyrimidine. This TFO can inhibit the expression of HBV genes and the replication of the virus. It includes two types of TFO. One can bind to two fragments of homologous homopolypurine/homopolypyrimidine in the DR region of HBV. The other binds to two fragments of homologous homopolypurine/homopolypyrimidine in the promoter region of the pre-S gene of HBV adr subtype. Their stability can also be increased by 3xe2x80x2-monophosphorylation or other chemical modifications. These TFOs can be used as therapeutic agents in the treatment of hepatitis B.
The invention provides a type of TFO which is able to form a triplex DNA structure with two fragments of homologous homopolypurine/homopolypyrimidine. The invention is based on the mechanism of triplex DNA formation and the sequences in HBV genes. It is aimed at two fragments of homologous homopolypurine/homopolypyrimidine sequences in the DR region of HBV and the promoter region of pre-S gene of HBV adr subtype. The invention involves the corresponding TFOs, as well as the synthesis on a DNA synthesiser of these TFOs and 3xe2x80x2-monophosphorylated TFO derivatives (see FIG. 1 and FIG. 2):
In order to study the binding ability of TFO to the two fragments of homologous homopolypurine/homopolypyrimidine in the DR region of HBV and the promoter region of pre-S gene of HBV adr subtype, the thermodynamic parameters of triplex DNA formation were measured using band mobility shift assays. TFOs B1-B5 can bind to the two fragments of homologous homopolypurine/homopolypyrimidine in pre-S genes of HBV. Of these, B1-B4 bind more strongly than B5, and B4 showed the strongest binding activity. TFOs B11, B12 and B15 can bind to the two fragments of homologous homopolypurine/homopolypyrimidine in the DR region of HBV. Of these, the binding activity of B15 and B12 is stronger than B11, and B15 is the strongest. B5 and B11 can only bind one fragment of homologous homopolypurine/homopolypyrimidine in the promoter region of the pre-S gene of HBV adr subtype and the DR region of HBV, respectively. The binding activity of TFOs B1-B4is stronger than B5, which explains the fact that TFOs B1-B4 can bind the two fragments of homologous homopolypurine/homopolypyrimidine in the pre-S gene of HBV.
TFO B6 is another oligonucleotide example which proves the point. In contrast with TFO B5, TFO B6 has extra eight Ts at the 3xe2x80x2-end, and can not bind to the second fragment of homologous homopolypurine/homopolypyrimidine. Also, it binds target sequences much more weakly than B5. Likewise, the binding activities of TFOs B15 and B12 are stronger than B11, which indicates that TFOs B15 and B12 can bind to the two fragments of homologous homopolypurine/homopolypyrimidine in the DR region of HBV. These results show that the TFOs disclosed in this invention can bind two fragments of homologous homopolypurine/homopolypyrimidine to form a new triplex DNA structure. Moreover, the bases in the TFO with a strong matching capability bind the target DNA by Hoogsteen bonds or anti-Hoogsteen bonds. When the new TFOs described in this invention bind the two fragments of homologous homopolypurine/homopolypyrimidine in the DR region of HBV and the promoter region of pre-S gene of HBV adr subtype, the target dsDNA structure changes from purines to pyrimidines. Although these changes reduce the stability of triplex DNA, the stability and specificity of the binding between the extended TFO and target DNA ultimately undergoes a major increase. Thus they show stronger inhibition. This also proves that the major factor affecting the stability of triplex DNA formation is the length of the TFO.
In order to study TFO""s inhibition of HBV gene expression and the inhibition of viral replication, hepatic HepG2 cells transfected by a plasmid (p1.2II) containing HBV genes were used as a model for studying the effects of TFO. Plasmid (p1.2II) contains cloned HBV (adr subtype) genes 1.2 times normal length, including the full length of the HBV genome and the overlapping region 1403-1983, and which can express all of the HBV mRNA. After p1.2II transfection of HepG2 cells, intact and infectious viral particles were produced. The inhibition of HBV genes by TFO was observed using commercial kits to test the expression levels of HBsAg and HBeAg. The inhibition of HBV replication by TFO was observed by measurement of the number of copies of HBV DNA in the cells.
Given that serum and cells contain high nuclease activity, TFOs can be chemically modified so as to increase their stability. Examples of modification are thiophosphorylation, methyl-phosphorylation, 2xe2x80x2-O-methylation and 3xe2x80x2-monophosphorylation, etc. Serum mostly contains 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity, and this enzyme requires a nucleic acid with a 3xe2x80x2-OH end as a substrate. According to previous results of the inventor (refer to patent no. 97106495.4, application date: Jun. 28, 1997), 3xe2x80x2-monophosphorylated oligonucleotides proved to be the best derivative. After the 3xe2x80x2-OH of an oligonucleotide was phosphorylated, it could no longer serve as a substrate for the 3xe2x80x2xe2x86x925xe2x80x2 exonuclease, thereby prolonging its retention in serum and cells. Thus it could more efficiently bind target genes. The stability of 3xe2x80x2-phosphorylated oligonucleotides in serum is markedly higher than that of unmodified oligonucleotides, and is slightly higher than for thiophosphorylated oligonucleotides. The uptake of 3xe2x80x2-phosphorylated oligonucleotides by cells is also higher than for unmodified or thiophosphorylated oligonucleotides. In addition, as 3xe2x80x2-phosphorylated oligonucleotides do not carry any unnatural components in the modification, its metabolites exhibit no toxicity or side-effects. Therefore, 3xe2x80x2-phosphorylation is an even better modification method, being superior and safer.
TFOs B4(3xe2x80x2P) and B15(3xe2x80x2P), which exhibit the strongest binding to target sequences, were selected for studying the ability of TFO to inhibit the expression of HBV genes and to inhibit the replication of HBV. The results show that the TFOs synthesised in this study inhibited the expression of HBV genes and the replication of HBV DNA, thereby inhibiting the replication of hepatitis B virus. Hence, these TFOs can be used to produce drugs for the inhibition of HBV and the treatment of hepatitis B.
In addition, HBV is a type of virus in which the genes are closely packed together. Four promoter regions of the virus genes all overlap with coding regions. Moreover, the transcription products, four mRNA sequences of 3.5 kb, 2.4 kb, 2.1 kb and 0.8 kb length respectively, all share a comnmon 3xe2x80x2-end. The 3xe2x80x2-end of a 3.5 kb pre-gene RNA is the template for reverse transcription of the virus into DNA. The two target sequences disclosed by the invention are both located in the 3.5 kb pre-gene RNA. The DR region is in the 3xe2x80x2-end of the 3.5 kb pre-gene RNA and the pre-S gene region is in the middle of the pre-gene RNA. At the same time, adding the anti-sense sequence oligonucleotide of the DR region: AsDR 5xe2x80x2 TCT CCT CCC CCA ACT CCT CCC 3xe2x80x2 (SEQ ID NO: 11) or its derivatives formed (DNA)2:RNA heterotriplex with TFOs B15 (or B12, B11) and their derivatives by binding to target sequences in RNA. Alternatively, adding the anti-sense sequence oligonucleotide of the pre-S region: AsPS 5xe2x80x2 GGA GGC AGG AGG AGG AA 3xe2x80x2 (SEQ ID NO: 12) or its derivatives formed (DNA)2:RNA heterotriplex with TFOs B4 (or B1, B2, B3, B5) and their derivatives by binding to target sequences in RNA. Also, adding the anti-sense sequence oligonucleotide of the DR region: AsDR (3xe2x80x2P) 5xe2x80x2 TCT CCT CCC CCA ACT CCT CCCp 3xe2x80x2 (SEQ ID NO: 11) formed (DNA)2:RNA heterotriplex with TFOs B15 (3xe2x80x2P) for B12 (3xe2x80x2P), Eu (3xe2x80x2P)] by binding to target sequences of RNA. Or, adding the anti-sense sequence oligonucleotide of the pre-S region: AsPS (3xe2x80x2P) 5xe2x80x2 GGA GGC AGG AGG AGG AAp 3xe2x80x2 (SEQ ID NO: 12) formed (DNA)2:DNA [sic] heterotriplex with TFOs B4 (3xe2x80x2P) [or B1 (3xe2x80x2P), B2 (3xe2x80x2P), B5 (3xe2x80x2P)]. This segment of (DNA)2:RNA heterotriplex sequence inhibited reverse transcription of virus into DNA by blocking the movement of the replicating machinery. This increased the effect of TFO in inhibiting the hepatitis B virus.
Thus, combining TFOs or TFO derivatives with the anti-sense sequence oligonucleotide of the same region not only inhibits transcription of HBV genes, but it can also form heterotriplexes in the target sequence of pre-RNA, which inhibits DNA reverse transcription. The end result is the more effective inhibition of HBV reproduction. Thus, TFO and its derivatives B4 (or B1, B2, B3, and B5) combined with AsPS can be used to make drugs for the inhibition of HBV and the treatment of Hepatitis B. Similarly, B15 (or B11, B12) combined with AsDR can also be used, as can the TFOs and their derivatives B4(3xe2x80x2P) [or B1(3xe2x80x2P), B2(3xe2x80x2P), B3(3xe2x80x2P), B5(3xe2x80x2P)] with ASPS (3xe2x80x2P), as well as B15(3xe2x80x2P) [or B11(3xe2x80x2P), B12(3xe2x80x2P)] with AsDR (3xe2x80x2P).
1. The invention provides a series of TFOs and their derivatives which form triplex DNA with the two fragments of homologous homopolypurine/homopolypyrimidine. The stability and specificity of the triplex DNA are increased by the extension of oligonucleotide length. As the TFOs form triplex DNA with the two fragments of homologous homopolypurine/homopolypyrimidine, the applicability of TFO in the Antigen Strategy is extended.
2. The invention provides a series of TFOs which can inhibit the expression of HBV genes and the reproduction of HBV. This represents a material which can directly inhibit the expression of HBV genes and the reproduction of HBV, and thereby provides a new therapeutic approach for hepatitis B.
3. The inhibition by TFO of HBV gene expression and HBV reproduction are highly specific and do not of themselves affect human cells.
4. This invention provides increased in vivo stability of the TFOs by 3xe2x80x2-monophosphorylation, which also markedly increases their inhibition activity. As the TFOs described in this invention do not contain any artificial components, their metabolites have no toxic side-effects in humans.
5. At the same time, adding an anti-sense oligonucleotide derived from the same region, allows it to combine with a TFO, bind target sequence RNA and form a (DNA)2:RNA heterotriplex structure, which blocks the movement of the replication machinery, thereby inhibiting virus RNA reverse transcription into DNA, and increasing the anti-HBV effect of TFO.